技术资料

The recent Zika virus (ZIKV) epidemic is associated with microcephaly in newborns. Although the connection between ZIKV and neurodevelopmental defects is widely recognized,the underlying mechanisms are poorly understood. Here we show that two recently isolated strains of ZIKV,an American strain from an infected fetal brain (FB-GWUH-2016) and a closely-related Asian strain (H/PF/2013),productively infect human iPSC-derived brain organoids. Both of these strains readily target to and replicate in proliferating ventricular zone (VZ) apical progenitors. The main phenotypic effect was premature differentiation of neural progenitors associated with centrosome perturbation,even during early stages of infection,leading to progenitor depletion,disruption of the VZ,impaired neurogenesis,and cortical thinning. The infection pattern and cellular outcome differ from those seen with the extensively passaged ZIKV strain MR766. The structural changes we see after infection with these more recently isolated viral strains closely resemble those seen in ZIKV-associated microcephaly. View Publication

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and,therefore,are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex,a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover,increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality. View Publication

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However,most iPSC lines are derived in advance of,and without the benefit of,neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants,these methods require outgrowth of cadaver tissue,which require additional time and is often only successul 50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue,iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases. View Publication

BACKGROUND Fragile X syndrome (FXS),a common cause of intellectual disability and autism,results from the expansion of a CGG-repeat tract in the 5' untranslated region of the FMR1 gene to<200 repeats. Such expanded alleles,known as full mutation (FM) alleles,are epigenetically silenced in differentiated cells thus resulting in the loss of FMRP,a protein important for learning and memory. The timing of repeat expansion and FMR1 gene silencing is controversial. METHODS We monitored the repeat size and methylation status of FMR1 alleles with expanded CGG repeats in patient-derived induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) that were grown for extended period of time either as stem cells or differentiated into neurons. We used a PCR assay optimized for the amplification of large CGG repeats for sizing,and a quantitative methylation-specific PCR for the analysis of FMR1 promoter methylation. The FMR1 mRNA levels were analyzed by qRT-PCR. FMRP levels were determined by western blotting and immunofluorescence. Chromatin immunoprecipitation was used to study the association of repressive histone marks with the FMR1 gene in FXS ESCs. RESULTS We show here that while FMR1 gene silencing can be seen in FXS embryonic stem cells (ESCs),some silenced alleles contract and when the repeat number drops below ˜400,DNA methylation erodes,even when the repeat number remains<200. The resultant active alleles do not show the large step-wise expansions seen in stem cells from other repeat expansion diseases. Furthermore,there may be selection against large active alleles and these alleles do not expand further or become silenced on neuronal differentiation. CONCLUSIONS Our data support the hypotheses that (i) large expansions occur prezygotically or in the very early embryo,(ii) large unmethylated alleles may be deleterious in stem cells,(iii) methylation can occur on alleles with<400 repeats very early in embryogenesis,and (iv) expansion and contraction may occur by different mechanisms. Our data also suggest that the threshold for stable methylation of FM alleles may be higher than previously thought. A higher threshold might explain why some carriers of FM alleles escape methylation. It may also provide a simple explanation for why silencing has not been observed in mouse models with<200 repeats. View Publication

Adverse effect of alcohol on neural function has been well documented. Especially,the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models,which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described,the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation,Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway,neuroactive ligand-receptor interaction,Toll-like receptor (TLR) signaling pathway,cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3,which is associated with nociception,a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs,but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response. View Publication

Pluripotent stem cells can become any cell type found in the body. Accordingly,one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells,which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next,we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture,hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells. View Publication

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short,enzymatically biotinylated tag,compatible with SRI techniques including uPAINT,STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues,with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β,neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody,and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore,Nlg1 is dynamic,disperse and sensitive to synaptic stimulation,whereas LRRTM2 is organized in compact and stable nanodomains. Thus,mSA is a versatile tool to image membrane proteins at high resolution in complex live environments,providing novel information about the nano-organization of biological structures. View Publication

Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression,especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies,including in vitro modeling of PD. However,further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs. View Publication

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons,and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts,whereas 30 proteins were increased in SMA motor neurons vs. their fibroblasts. Notably,UBA1 was significantly decreased in SMA motor neurons,supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons,including beta III-tubulin and UCHL1,were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts,highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons,and for the identification of novel biomarker and therapeutic targets for SMA. View Publication

Synaptic plasticity is mediated by the dynamic localization of proteins to and from synapses. This is controlled,in part,through activity-induced palmitoylation of synaptic proteins. Here we report that the ability of the palmitoyl-acyl transferase,DHHC5,to palmitoylate substrates in an activity-dependent manner is dependent on changes in its subcellular localization. Under basal conditions,DHHC5 is bound to PSD-95 and Fyn kinase,and is stabilized at the synaptic membrane through Fyn-mediated phosphorylation of a tyrosine residue within the endocytic motif of DHHC5. In contrast,DHHC5's substrate,δ-catenin,is highly localized to dendritic shafts,resulting in the segregation of the enzyme/substrate pair. Neuronal activity disrupts DHHC5/PSD-95/Fyn kinase complexes,enhancing DHHC5 endocytosis,its translocation to dendritic shafts and its association with δ-catenin. Following DHHC5-mediated palmitoylation of δ-catenin,DHHC5 and δ-catenin are trafficked together back into spines where δ-catenin increases cadherin stabilization and recruitment of AMPA receptors to the synaptic membrane. View Publication

For diseases of the brain,the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus,POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro,retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages,and responded to retinoic acid,promoting a more posterior fate (HOXB4+,OTX2-,and GBX2-). These findings offer insight into pig iPSC development,which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models,providing relevant translational data for eventual human neural cell therapies. View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication